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A common activity in the global search for useful Cry toxins is the microscopic screening of bacterial colonies for the presence of Bacillus thuringiensis. High-throughput screens require that aliquots from large numbers of colonies be arrayed on a microscopic slide. However, precisely placing a small amount of bacteria on a slide, and at a density that is useful for microscopic examination, is both difficult to achieve and time consuming. Herein we share a simple technique that utilizes a hooked wand and small polystyrene beads to quickly collect, and uniformly apply, aliquots of bacterial colonies onto gridded microscope slides in a manner optimal for viewing. If desired, libraries of examined bacteria can simultaneously be generated by discharging the beads into indexed multiwell plates. This simple and inexpensive method is robust, suitable for both light and phase contrast microscopy, and has been also used successfully to screen randomly mutated bacteria for phenotypic changes.

Parasporins represent a new functional class of Cry (crystal protein) toxins produced by the bacterium Bacillus thuringiensis (Bt). Unlike Cry toxins that demonstrate activity mainly against some insect cells, parasporins are characterized as being non-hemolytic, yet capable of preferentially killing some human cancer cells. Globally, six different parasporin types, PS1-PS6, based on protein sequence homology, have been identified in only four countries (Japan, Vietnam, India, and Canada). Herein we report the results of a screening study of 160 Bt isolates collected from the Caribbean island of Trinidad. One isolate (strain 64-1-94) was shown to kill human cancer cells and to contain one ps6 and two ps1 parasporin genes. The two ps1 genes were located approximately 6 kb apart from each other, sharing a similar spatial arrangement, and high sequence homology, with two plasmid-located ps1 genes, ps1Aa6 and ps1Ad1, recently isolated from a Japanese strain. Evidence is also presented that a parasporin gene reported previously for a Canadian strain, ps1Aa2, is most likely derived from a recombination event between these same two genes found in the Trinidadian and Japanese strains. Notably, all three strains share a ps6 parasporin gene, presumably located on a separate plasmid. These data suggest that the global population of ps1 genes may be have originated from a single pair of parasporin genes. Given the large geographical distance between the collection sites, which are located on both continental land masses and islands at sea, ps1 genes are able to retain a remarkable level of homology not easily explained.

Bacillus thuringiensis is a bacterium best known for its production of crystal-like bodies comprised of one or more Cry-proteins, which can be toxic to insects, nematodes or cancer cells. Although strains of B. thuringiensis have occasionally been observed with filamentous appendages attached to their spores, appendages in association with their parasporal bodies are extremely rare. Herein we report the characterization of Bt1-88, a bacterial strain isolated from the Caribbean that produces a spore-crystal complex containing six long appendages, each comprised of numerous thinner filaments approximately 10 nm in diameter and 2.5 μm in length. Each of the multi-filament appendages was attached to a single, small parasporal body located at one end of the bacterial spore. Biochemical tests, 16S rDNA gene sequencing, and the identification of two Cry proteins by partial protein sequencing (putatively Cry1A and Cry2A), unambiguously identified Bt1-88 as a strain of B. thuringiensis. Bt1-88 represents the second reported strain of B. thuringiensis possessing a parasporal body/appendage phenotype characterized by one or more long appendages, comprised of numerous filaments in association with a parasporal body. This finding suggests that Bt1-88 is a member of a new phenotypic class of B. thuringiensis, in which the parasporal body may perform a novel structural role through its association with multi-filament appendages.

We report on a novel bacterium, isolated during a screen for environmental isolates of Bacillus thuringiensis, that possesses a novel filamentous structure. Nucleotide sequence from the isolate’s 16S rRNA gene places the bacterium unambiguously within the Bacillus thuringiensis/Bacillus cereus group. Phase-contrast and electron microscopy indicate the presence of both a parasporal body and a long filament which are retained after sporulation. The filament is shown to consistently arise from the end of the exosporium and next to the parasporal body. Upon spore germination, the parasporal body/filament complex is retained on the cell wall of the resulting bacterium.

We report on the construction of a novel strain of E. coli that can be useful for studies on the structure/function relationship of 5S rRNAs. The bacterial strain is deficient in six of the eight naturally occurring 5S rRNA genes (operons B, D, H, G, E) and demonstrates a greatly reduced growth rate that can be compensated by the plasmid-encoded expression of 5S rRNA. The relatively large difference in growth rate between compensated and non-compensated mutants provides the basis for a quick and simple assaying system for both the evaluation and mass screening of divergent 5S rRNA sequences for function. We describe the construction of the 5S rRNA deletion mutant BDHGE and characterize the usefulness and limitations of the system for evaluating structure/function relationships of 5S rRNA sequence.

Contamination of the environment with human sewage represents a serious public health concern in which Escherichia coli plays a central role, either directly as a human pathogen or indirectly through its use as an indicator organism. There is thus an ongoing effort to better understand the behavior of E. coli within such environments. Useful to such studies is the ability to readily detect a specific E. coli population and distinguish it from similar indigenous bacteria. Herein, we report the construction of an E. coli strain (PCPHR) that expresses a Stable Artificial RNA (SAR) from the chromosomal rrnH operon. The SAR product is present in large numbers of copies/cell and thus provides an enhanced detection signal without significant effect on the wild-type growth rate. Detection can be accomplished by any of several routine molecular methods. Preliminary studies suggest SAR expression levels correlate positively with growth. PCPHR is immediately available for use as a marker strain for E. coli in application in the arena of public health or environmental studies.